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Probe

Imagen ® One-Step Probe RT-PCR Fastermix

Cat. No IPRF 001

Description

Imagen ® One-Step Probe RT-PCR Fastermix is a ready-to-use and that can perform cDNA synthesis and PCR reaction all at once, 2 X concentration premix including a novel Fast Hotstart Taq DNA polymerase, Fast Reverse transcriptase, optimized reaction buffer and nucleotides with combines accurate using sequence-specific fluorogenic probe chemistries including hydrolysis probes (ex. TaqMan, Molecular beacon, etc…). This formulation is engineered for superior efficiency in the conversion of sample RNA into cDNA and sensitive amplification.

Contents

The Fastermix is supplied as a ready-to-use 2x reaction

mix. The formulation contains reverse transcriptase, DNA

polymerase enzyme, dNTPs, MgCl₂, reaction enhancers,

and stabilizers.

Storage

-20 °C

Protected from light

Avoid repeated freezing and throwing

Reaction Mix Thawing and Handling

The Imagen ® One-Step Probe RT-PCR Fastermix is delivered in a 2x ready-to-use format. To use the mix, thaw the vial on ice to 4 °C.

Please completely mix the vial and briefly centrifuge to ensure all components are at the bottom of the tube. Store on ice protected from light until ready to use. If using automated liquid handling, let sit at ambient temperature for 10 min to further reduce the viscosity.

Application

One step qRT-PCR based on Specific Probes.

Detection and Quantification of DNA and cDNA targets.

Gene Expression.

Fast qPCR platforms.

High Throughput Applications.

Recommend for Assay Design and Optimization

For best qPCR efficiency, design assays targeting an amplicon size of 70 - 150bp

The Imagen ® One-Step Probe RT-PCR Fastermix cycling protocols have been optimized for assays with a predicted primer melting temperature (Tm) of 58 - 62°C.

The probe’s Tm should be 8 – 10 °C higher than the calculated primer Tm.

Prepare the RT-PCR Reaction Mix

Mix the Imagen ® One-Step Probe RT-PCR Fastermix thoroughly but gently until it’s completely homogenous.

Prepare the RT-PCR Reaction Mix for the number of reactions required as shown in table below and plus 10% overage.

Reagent

Volume (ul)

Final conc.

2 x One-Step Probe RT-PCR Fastermix

12.5

1x

Forward Primer

Variable

300 - 600 nM

Reverse Primer

Variable

300 - 600 nM

Fluorogenic Probe

Variable

200 – 200nM

RNA Template

2 - 4

100 ng - 10 pg

Nuclease-free water

Up to 25

-

Final volume

25

-

Vortex the tube to mix the contents thoroughly, then centrifuge briefly to collect the contents at the bottom of the tube. (*Use good pipetting practice to ensure assay precision and accuracy of dispensing.)

Add RNA (and nuclease-free water, if needed) to the PCR tubes or wells containing the reaction mix, seal　tubes or wells with flat caps or optically transparent film, and gently vortex to ensure thorough mixing of the reaction components.

Program the thermal cycling protocol on the real-time PCR instrument.

Step

Temp. ℃

Time

Cycles

Reverse transcription

45 – 48

5 - 10 min

1

DNA polymerase activation and template denaturation

95

1 min

1

Amplification

Template denaturation

95

3 sec

40

Annealing / Extension and plate read

58 - 62

≧20 sec

Load the PCR tubes or plates onto the real-time PCR instrument and start the RT-PCR run program.

When thermal cycling is complete, perform data according to the instructions in the instrument-specific software.